Doctor of Philosophy
School of Biological Sciences - Faculty of Science
Wyatt, Amy Ruth, The role of clusterin (CLU) in extracellular protein folding quality control, PhD thesis, School of Biological Sciences, University of Wollongong, 2009. http://ro.uow.edu.au/theses/860
Processes to attain and maintain the correct three-dimensional shape, known as the native conformation, of proteins are vital. However, certain conditions including thermal and oxidative stress may cause proteins to partially unfold and aggregate. Intracellular and/or extracellular protein aggregates have been identified in a large number of diseases, including Alzheimer’s disease, arthritis and type II diabetes. While intracellular quality control for the folding state of proteins is well characterized, corresponding mechanisms for extracellular protein folding quality control have yet to be described.
Clusterin (CLU) is an abundant extracellular chaperone that can stabilize proteins and prevent their precipitation during exposure to elevated temperatures or oxidative stress in vitro. The work described here demonstrates that CLU stabilizes stressed client proteins by forming soluble, high molecular weight (HMW) complexes with them. The maximum loading of CLU appears to be at a mass ratio of CLU:stressed client protein of approximately 1:2 (irrespective of the identity of the client protein or the temperature used to induce heat stress). It was demonstrated that various human plasma proteins show increased association with CLU after plasma is subjected to mild shear stress or oxidative stress at 37°C - the most abundant of these was fibrinogen (FGN) which co-purified with CLU from stressed plasma. In vitro, using purified proteins, heat stress of 45°C for 12 h was required to induce FGN precipitation and the formation of HMW CLU-FGN complexes. Size exclusion chromatography (SEC) of the stressed plasma suggested that a portion of the complexes formed in plasma between CLU and FGN may be similar in mass to those formed in vitro.
Using surface plasmon resonance, although CLU was found to bind to megalin, only minimal (or no) binding of HMW complexes formed between CLU and glutathione-S-transferase, citrate synthase or lysozyme was detected. Similarly, negligible binding of these complexes to low density lipoprotein receptor superfamily members expressed on the surface of the rat yolk sac cell line BN was detected. However, the complexes were shown to preferentially bind to the surface of BN cells, peripheral human monocytes and rat hepatocytes (more so than uncomplexed CLU or client proteins). In all cases, this binding was inhibited by fucoidin (a scavenger receptor inhibitor). Confocal microscopy suggested that binding of HMW CLU-stressed protein complexes to the surface of BN cells or rat hepatocytes was followed by their internalization into lysosomes. Furthermore, Western blotting showed that hepatocytes were able to degrade the HMW CLU-stressed protein complexes and that the degradation was almost completely abolished by inhibiting lysosomal proteases with chloroquine. The results of in vivo biodistribution studies in Sprague Dawley rats were highly consistent for several different HMW CLU-stressed protein complexes. Intravenous 123I-labelled HMW CLU-stressed protein complexes were cleared more efficiently from circulation compared to free CLU and the uncomplexed client proteins. The liver and to a lesser degree the spleen appeared to be the key organs responsible for the uptake of complexes and this uptake was inhibited by pre-injection of the animals with fucoidin.
The findings of this study suggest an important role for CLU in global quality control of extracellular protein folding. It appears likely that stressed (partially unfolded) extracellular proteins are stabilized and held in solution by CLU and that CLU-stressed protein complexes are subsequently taken up by fucoidin-inhibitable cell surface receptors for subsequent degradation within lysosomes. The precise physical characteristic or binding site that targets CLU-stressed protein complexes for receptor mediated uptake remains to be identified and further work is needed to determine the particular receptor(s) involved. The findings of this study support a model in which complexation with CLU is an important first step in the targeted disposal of stressed proteins via scavenger-like endocytic receptors.
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